CN104745597A - Method for efficiently expressing recombinant liraglutide - Google Patents
Method for efficiently expressing recombinant liraglutide Download PDFInfo
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- CN104745597A CN104745597A CN201510140642.6A CN201510140642A CN104745597A CN 104745597 A CN104745597 A CN 104745597A CN 201510140642 A CN201510140642 A CN 201510140642A CN 104745597 A CN104745597 A CN 104745597A
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Abstract
The invention relates to the biomedical field, and in particular relates to a method for efficiently expressing recombinant liraglutide. The method for efficiently expressing recombinant liraglutide is used for performing gene cloning and transformed expression. The recombinant liraglutide is constructed, and liraglutide protein is prepared through an escherichia coli expression technology. Compared with the two methods, the method disclosed by the invention is simple and convenient to operate, high in yield and low in cost.
Description
Technical field
The present invention relates to biomedicine field, particularly relate to the method for a kind of high expression restructuring Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37].
Background technology
Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] is a kind of people's glicentin-1 (GLP-1) analogue, is used for the treatment of diabetes.
Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] behaviour glicentin-1 (GLP-1) analogue, an amino acid difference is had compared with natural GLP-1 molecular structure, and add 16 carbon palmityl fatty acid side chains, but there is 95% homology with natural human GLP-1, remain effect of natural GLP-1, it can in conjunction with and activate GLP-1 acceptor, promote pancreatic beta cell glucose concn dependency ground excreting insulin, the pattern simultaneously relied on glucose concn reduces the secretion of too high glicentin.The Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] injection developed by Novo Nordisk Co., Ltd be proved treatment type II diabetes in there is good result, but need every day drug administration by injection once, make patient's conformability poor.
Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] peptide sequence is:
H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Le u-Glu-Gly-Gln-Ala-Ala-Lys (N-ε-(N-α-Palmitoyl-L-γ-glutamyl))-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-OH Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] to be first of being developed by Novo Nordisk Co., Ltd of Denmark be also a unique long-acting GLP-1 analogue at present, there is the effect of GLP-1 receptor stimulant, similar to GLP-1 in molecular structure, biological activity, action target spot and immunogenicity etc.The homology of the molecular structure of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] and GLP-1 (7-37) reaches 97%, textural difference shows that Lys 34 is substituted by Arg, Lys 26 is via glutamate-induced generation palmitoylation, fatty acid side chain can make Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] reversibly be combined with albumin in blood night, make the extended durations of action of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], and the opposing strengthened DPP-4 enzyme liberating, fatty acid side chain can also make Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] molecule, and in injection site, selfing is unified into heptamer, thus delay it from subcutaneous attraction, make to be its action time close to 24 hours, every day injects once and can inject at any time, and hypoglycemia occurrence risk is little.The Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] of Novo Nordisk is prepared by biological methods such as genetically engineereds, and technical difficulty is large, and production cost is high, is unfavorable for the scale operation of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37].
Summary of the invention
In order to solve above-mentioned technical problem, the first object of the present invention is to provide the expressing gene fragment HIS of a kind of high expression restructuring Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]
6-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], second object of the present invention is to provide the method for a kind of high expression restructuring Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], and the method is undertaken recombinant expressed by biological methods such as genetically engineereds, reduces production cost, pollute and reduce, be conducive to the scale operation of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37].
In order to realize first above-mentioned object, present invention employs following technical scheme:
A kind of expressing gene fragment HIS of high expression restructuring Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]
6-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], the sequence of this expressing gene fragment is as shown in SEQ ID NO:1.
The invention also discloses the recombinant expression vector containing above-mentioned expressing gene fragment.
As preferably, this recombinant expression vector is that described gene fragment clone enters in PET-22b carrier and obtains recombinant expression vector PET-22b-HIS
6-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37].
As preferably, this recombinant expression vector adopts and molecular chaperones troponin C SEQ ID NO:2 full-length gene is connected to described gene fragment HIS
6the 5` end of-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], eventually clone enters pET-22b carrier, is namely reassembled as expression vector pET-22b-troponin C-HIS
6-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37].
The invention also discloses the bacterial strain containing above-mentioned expressing gene fragment, this bacterial strain adopts expression vector pET-22b-troponin C-HIS
6-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] is transferred in intestinal bacteria DE3 bacterial strain, with the expression of IPTG liquid storage induction recombination, and collected by centrifugation thalline.
Above-mentioned bacterial strain may be used for the high expression of restructuring Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37].
In order to realize second above-mentioned object, present invention employs following technical scheme:
A method for high expression restructuring Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], the method comprises the following steps:
1) according to Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] gene sequence design primer, and the codon replacing the unfavorable expression of intestinal bacteria is to obtain the gene order optimized, gene order 5` end increases by two fragment gene sequences successively, is respectively intestines shock protein restriction enzyme site sequence EK and HIS sequence label HIS
6, obtain expressing gene fragment HIS
6-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], expressing gene fragment HIS
6the sequence of-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] is as shown in SEQ ID NO:1; And gene fragment clone is entered in PET-22b carrier be recombinant expression vector PET-22b-HIS
6-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], the corresponding recombinant protein given expression to is PET-22b-HIS
6-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]; Then again molecular chaperones troponin C SEQ ID NO:2 full-length gene order is cloned out, be then connected to gene fragment HIS
6the 5` end of-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], eventually clone enters pET-22b carrier, is namely reassembled as expression vector pET-22b-troponin C-HIS
6-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], the corresponding recombinant protein given expression to is troponin C-HIS
6-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37];
2) by step 1) in the expression vector pET-22b-troponin C-HIS that builds
6-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] is transferred in intestinal bacteria DE3 bacterial strain, with the expression of IPTG liquid storage induction recombination, and collected by centrifugation thalline; The resuspended thalline obtained of PB damping fluid, ultrasonication, collected by centrifugation supernatant liquor;
3) by 2) in the troponin C-HIS that obtains
6-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] albumen carries out preliminary purification, carries out purifying by Zeo-karb, and damping fluid is PB, 20mM, PH7.5, by troponin C-HIS
6-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] albumen salt ionic concentration wash-out is also collected;
4) target protein that collection wash-out obtains is carried out nickel ion affinity chromatograph, by HIS label, target protein is adsorbed on nickel ion affinity column, then carries out wash-out with high density imidazoles;
5) eluted protein is carried out desalination, balance liquid is 20mM PB; Cut with EK enzyme enzyme, 4 DEG C of enzymes that spend the night are cut;
6) finally by digestion products albumen through affinity chromatography, damping fluid is the same, is separated by fusion rotein with Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] albumen, stream wear for the purpose of albumen, wash-out be foreign protein.
In order to obtain the completely the same Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] albumen with sequence, the present invention utilizes intestines shock protein enzyme to remove label and the molecular chaperones troponin C of HIS6, expose the aminoterminal that it is original, after entering human body like this, can not antibody be produced, specific practice be desalination is obtained containing troponin C-HIS
6it is 6.0 that the solution of-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] albumen adjusts to pH, adds the further enzyme of intestines shock protein enzyme and cuts, finally discharge the on all four albumen Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] protein sequence.
The present invention solves how to carry out intestinal bacteria effectively expressing by genetic engineering technique method, carries out gene clone, transforms and expresses.By building restructuring Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] bacterial strain, prepare Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] albumen by escherichia coli expression technology, easy and simple to handle with kind of the Measures compare of two above, output is high, and cost is low.
Accompanying drawing explanation
Fig. 1 is the gene constructed plasmid vector of restructuring Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37].
Fig. 2 is Fig. 1 magnified partial view.
Fig. 3 is Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] protein electrophoresis SDS-PAGE.
Embodiment
The clone of A, coding Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] gene
According to Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] gene sequence design primer, and the codon replacing the unfavorable expression of intestinal bacteria is to obtain the gene order optimized, gene order 5` end increases by two fragment gene sequences successively, be respectively intestines shock protein restriction enzyme site sequence EK and HIS sequence label HIS6, obtain expressing gene segment HIS6-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], sequence is as shown in SEQ ID NO:1.
B, expression vector establishment
Then clone out by molecular chaperones troponin C full-length gene order again, sequence, as shown in SEQ ID NO:2, is then connected to gene segment HIS
6the 5` end of-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], is connected by T4DNA ligase enzyme with gene segment with the plasmid cut through same enzyme, will connect product electricity consumption robin or 42 DEG C of heat shock method transformation of E. coli BL21, and namely be reassembled as expression vector pET-22b-troponin C-HIS
6-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], the corresponding recombinant protein given expression to is troponin C-HIS
6-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37].The plasmid vector built is as Fig. 1.
The expression and purification of C, restructuring Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] gene
Single fresh colony is inoculated in the LB nutrient solution containing 100ug/ml Amlicillin, 37 DEG C of shaking culture.Next day is inoculated in comparatively large vol nutrient solution with 2% inoculum size.37 DEG C are cultured to OD600 about 1, add IPTG induction, continue shaking culture 4 hours, centrifugal collecting cell.
The purifying of D, restructuring Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] gene
By collected by centrifugation thalline, with the resuspended thalline obtained of the PB damping fluid of 0.1M, ultrasonication, collected by centrifugation supernatant liquor; Supernatant is diluted 5 times, carry out purifying by Zeo-karb, damping fluid is PB, 20mM, PH7.5, by troponinC-HIS
6-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] albumen salt ionic concentration wash-out is also collected.Balance liquid is 20mM PB, PH7.5, and elutriant is 20mM PB, PH7.5,0.2M NaCl, and target protein is mainly in elutriant.Again eluted protein is carried out affinity chromatography, balance liquid is 20mM PB, 0.2M NaCl, 50mM imidazoles, PH7.5, and elutriant is 20mM PB, PH7.5,0.2M NaCl, 0.2M imidazoles, and PH7.5, target protein is mainly in elutriant.Eluted protein is carried out desalination, and balance liquid is 20mM PB, then is cut by albumen EK enzyme enzyme, and 4 DEG C of enzymes that spend the night are cut.Final protein is through affinity chromatography, and damping fluid is the same, is separated by fusion rotein with Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] albumen, stream wear for the purpose of albumen, wash-out be foreign protein.
The Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] protein electrophoresis SDS-PAGE obtained is as Fig. 3, and from left to right, 1 is the troponin C-HIS of purifying
6-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] sample, 2 cut sample for enzyme, and 3 is Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] sample.
SEQ ID NO：1
gaattccaccaccaccaccaccacgacgatgatgacaagcatgctgaagggacctttaccagtgatgtaagttcttatttggaaggccaagctgccaaggaattcattgcttggctggtgcgtggccgtggatgaggatcc
SEQ ID NO：2
Atggcgtcaatgacggaccagcaggcggaggcccgcgccttcctcagcgaggagatgattgctgagttcaaagctgcctttgacatgtgggatgcggacggaggtggggacatcagcaccaaggagttgggcacggtgatgaggatgctgggccagaaccccaccaaagaggagctggatgccatcatcgaggaggtggacgaggatggcagcggcaccatcgacttcgaggagttcctggtgatgatggtgcgccagatgaaagaggacgccaagggcaagtctgaggaggagctggccaactgcttccgcatcttcgacaagaacgctgatgggttcatcgacatcgaggagctgggtgagattctcagggccactggggagcacgtcatcgaggaggacatagaagacctcatgaaggattcagacaagaacaatgacggccgcattgacttcgatgagttcctgaagatgatggagggtgagctcggagaggctcatgaagcagaagaggtccacgaagaagctcatcatgaggaagctcaccatgcggaagctcatcatgaggaagctcatgctcatgcggaagaagtccatgaaccagaagaagcccctgaagaagaggagaagcccagaattaat
Claims (8)
1. the expressing gene fragment HIS of a high expression restructuring Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]
6-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], the sequence of this expressing gene fragment is as shown in SEQ ID NO:1.
2. the recombinant expression vector containing expressing gene fragment according to claim 1.
3. recombinant expression vector according to claim 2, is characterized in that this recombinant expression vector is that described gene fragment clone enters in PET-22b carrier and obtains recombinant expression vector PET-22b-HIS
6-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37].
4. recombinant expression vector according to claim 2, is characterized in that this recombinant expression vector adopts and molecular chaperones troponin C SEQ ID NO:2 full-length gene is connected to described gene fragment HIS
6the 5` end of-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], eventually clone enters pET-22b carrier, is namely reassembled as expression vector pET-22b-troponin C-HIS
6-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37].
5. the bacterial strain containing expressing gene fragment according to claim 1, is characterized in that: this bacterial strain adopts expression vector pET-22b-troponin C-HIS according to claim 4
6-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] is transferred in intestinal bacteria DE3 bacterial strain, with the expression of IPTG liquid storage induction recombination, and collected by centrifugation thalline.
6. the bacterial strain shown in claim 5 is for the high expression of Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] of recombinating.
7. a method for high expression restructuring Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], is characterized in that the method comprises the following steps:
1) according to Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] gene sequence design primer, and the codon replacing the unfavorable expression of intestinal bacteria is to obtain the gene order optimized, gene order 5` end increases by two fragment gene sequences successively, is respectively intestines shock protein restriction enzyme site sequence EK and HIS sequence label HIS
6, obtain expressing gene fragment HIS
6-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], expressing gene fragment HIS
6the sequence of-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] is as shown in SEQ ID NO:1; And gene fragment clone is entered in PET-22b carrier be recombinant expression vector PET-22b-HIS
6-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], the corresponding recombinant protein given expression to is PET-22b-HIS
6-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37]; Then again molecular chaperones troponin C SEQ ID NO:2 full-length gene order is cloned out, be then connected to gene fragment HIS
6the 5` end of-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], eventually clone enters pET-22b carrier, is namely reassembled as expression vector pET-22b-troponin C-HIS
6-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37], the corresponding recombinant protein given expression to is troponinC-HIS
6-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37];
2) by step 1) in the expression vector pET-22b-troponin C-HIS that builds
6-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] is transferred in intestinal bacteria DE3 bacterial strain, with the expression of IPTG liquid storage induction recombination, and collected by centrifugation thalline; The resuspended thalline obtained of PB damping fluid, ultrasonication, collected by centrifugation supernatant liquor;
3) by 2) in the troponin C-HIS that obtains
6-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] albumen carries out preliminary purification, carries out purifying by Zeo-karb, and damping fluid is PB, 20mM, PH7.5, by troponin C-HIS
6-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] albumen salt ionic concentration wash-out is also collected;
4) target protein that collection wash-out obtains is carried out nickel ion affinity chromatograph, by HIS label, target protein is adsorbed on nickel ion affinity column, then carries out wash-out with high density imidazoles;
5) eluted protein is carried out desalination, balance liquid is 20mM PB; Cut with EK enzyme enzyme, 4 DEG C of enzymes that spend the night are cut;
6) finally by digestion products albumen through affinity chromatography, damping fluid is the same, is separated by fusion rotein with Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] albumen, stream wear for the purpose of albumen, wash-out be foreign protein.
8. the method for a kind of high expression restructuring Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] according to claim 7, is characterized in that: what desalination obtained contains troponin C-HIS
6it is 6.0 that the solution of-EK-Arg34Lys26-(N-EPSILON-(N-ALPHA-Palmitoyl-L-GAMMA-glutamyl))-GLP-1[7-37] albumen adjusts to pH, adds the further enzyme of intestines shock protein enzyme and cuts.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2017021819A1 (en) * | 2015-07-31 | 2017-02-09 | Dr. Reddy’S Laboratories Limited | Process for preparation of protein or peptide |
| CN110092825A (en) * | 2019-05-06 | 2019-08-06 | 山东汉泰生物科技有限公司 | A method of Liraglutide intermediate polypeptide is prepared using genetic recombination |
| WO2019153827A1 (en) * | 2018-02-06 | 2019-08-15 | 美药星（南京）制药有限公司 | Method for preparing liraglutide intermediate polypeptide |
| WO2020127476A1 (en) | 2018-12-19 | 2020-06-25 | Krka, D.D., Novo Mesto | Pharmaceutical composition comprising glp-1 analogue |
| WO2021123228A1 (en) | 2019-12-18 | 2021-06-24 | Krka, D.D., Novo Mesto | Pharmaceutical composition comprising glp-1 analogue |
| WO2022012020A1 (en) * | 2020-07-17 | 2022-01-20 | 安徽新熙盟生物科技有限公司 | Preparation method for glp-1 analogue polypeptide and use thereof in type ii diabetes |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017021819A1 (en) * | 2015-07-31 | 2017-02-09 | Dr. Reddy’S Laboratories Limited | Process for preparation of protein or peptide |
| WO2019153827A1 (en) * | 2018-02-06 | 2019-08-15 | 美药星（南京）制药有限公司 | Method for preparing liraglutide intermediate polypeptide |
| US10851146B2 (en) | 2018-02-06 | 2020-12-01 | Amphastar Nanjing Pharmaceuticals Inc. | Method for preparing liraglutide intermediate polypeptide |
| WO2020127476A1 (en) | 2018-12-19 | 2020-06-25 | Krka, D.D., Novo Mesto | Pharmaceutical composition comprising glp-1 analogue |
| CN110092825A (en) * | 2019-05-06 | 2019-08-06 | 山东汉泰生物科技有限公司 | A method of Liraglutide intermediate polypeptide is prepared using genetic recombination |
| WO2021123228A1 (en) | 2019-12-18 | 2021-06-24 | Krka, D.D., Novo Mesto | Pharmaceutical composition comprising glp-1 analogue |
| WO2022012020A1 (en) * | 2020-07-17 | 2022-01-20 | 安徽新熙盟生物科技有限公司 | Preparation method for glp-1 analogue polypeptide and use thereof in type ii diabetes |
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